This application addresses broad Challenge Area Biomarker Discovery and Validation (03) and specific Challenge Topic Imaging Biomarkers (03-AR-104). We desire to detect important biomarkers associated with the onset of skin cancers, with a particular emphasis on melanoma. Specifically, we will apply a novel imaging technology (transient absorption microscopy) to image melanins and hemoglobins in developing skin lesions, in both fixed and live skin. The goals are to noninvasively detect melanomas developing in their earliest stages and to reduce false positives and false negatives in both dermoscopy and histopathology. Conventional melanoma detection and diagnosis is a two-tier process that begins with visual or dermoscopic inspection of suspicious moles and ends with the removal of suspected tissue that is examined in a microscope to confirm diagnosis. However, this approach faces two fundamental challenges. The first is the difficulty in visually detecting the differences between melanoma and benign moles;most melanomas are highly pigmented, and even using a dermoscope, doctors cannot see far beneath the surface. This problem is largely addressed by excision, H&E staining, and examination by a pathologist, although patients generally present far too many moles to excise and test in such an invasive manner. The second challenge is that false negatives and false positives from histopathology remain a serious problem even with trained observers. False negatives delay treatment and increase mortality;in fact misdiagnosis of melanoma is the second most common reason for cancer malpractice claims in the United States (after breast cancer). False positives drive up the cost of healthcare with unnecessary, expensive, and invasive procedures (including sentinel lymph node biopsy and systemic adjuvant therapy) and may make it impossible for the patient to obtain health or life insurance. Thus, more accurate detection and diagnosis could have a very large impact on patient survival and health care cost reduction. Existing microscopy methods are not well suited to deal with these challenges. Reflectance confocal scanning laser microscopy (rCSLM) has been commercialized for this application, but the contrast (scattering) suffers from a lack of specificity as it does not target a specific biomarker. Conventional multiphoton microscopy can image deep into tissue with microscopic resolution, but pigmented lesions present a horrible target because the generated light is reabsorbed (and in any event the fluorescence from melanin is extremely weak). Here we use nonlinear transient absorption microscopy, which does not retains the resolution advantage of multiphoton microscopy but does require the sample to generate light at a new wavelength. Such methods have been around for decades, but recent work in the PI's lab has exploited advanced femtosecond pulse shaping and pulse train modulation methods to dramatically increase the sensitivity- thus making it feasible to image tissue with modest powers. He and his research group have migrated this technology from basic science to clinical applications. The targeted biomarkers are significant for melanoma diagnosis. There is good evidence that the local ratio between pheomelanin and eumelanin contents are altered in developing melanoma;here we dramatically improve on previous work by measuring this distribution microscopically (in melanosomes and melanocytes), at depth in tissue, and noninvasively. There is also evidence that microvascularity and oxygenation (oxy- and deoxyhemoglobin, which we can also image noninvasively and at depth) correlates with metastatic potential. Overall, the research team in this project includes chemists, laser technologists, pathologists and dermatologists in order to truly gather the expertise needed to make a clinical impact. For example, the PI is a pioneer in molecular imaging and ultrafast laser physics (and Chair-Elect of the Division of Laser Science of the American Physical Society), but also a professor in Chemistry, Radiology and Biomedical Engineering. In the first aim, we focus on analyzing de-identified excised moles with our new methods and with the best conventional methods. As this work evolves, and we determine the molecular signatures which best correlate with cancer development, we will progress to freshly excised skin studies, in order to validate safe power limits (even though we currently use less laser power than existing commercial systems). Finally, we progress to a live animal model (human skin with induced lesions grafted on nude mice), where we will also measure blood flow and local tissue oxygenation as likely markers of aggressive metabolism. By the end of the grant period, we will be poised to provide spectacular and novel insight into the development of melanoma in vivo, and be ready for human clinical work. PUBLIC HEALTH RELEVANCE: We propose a novel imaging technology which can image specific cancer biomarkers in developing skin lesions, to noninvasively detect early melanomas without excision and to reduce false positives and false negatives in histopathology. Reducing false negatives would reduce cancer fatalities;reducing false positives reduces overall healthcare costs.